New Polymer Belts for Lipid-Bilayer Nanodiscs to Study the Mo-lecular Basis of GPCR Signalling – NanoBelt

Membrane proteins (MPs) play fundamental roles in biology because they control communication and material transfer within and between living cells and their environment. MPs account for about one third of all proteins encoded in the human genome and half of all drug targets. However, their extraction, purification, and in vitro investigation are very challenging. Current methods used for isolating MPs often depend on an inappropriate, overly aggressive chemistry that results in protein denaturation. This issue is particularly severe in the case of the numerous sensitive MPs that constitute important drug targets, as their poor stability upon removal from their native membrane environment seriously impairs the drug-discovery process.

Several new chemically inspired strategies have been explored to overcome this limitation. Among them, nanodiscs assembled from phospholipids and amphiphilic styrene/maleic acid (SMA) copolymers have received great attention. Unlike other systems, SMA can recruit MPs and lipids directly from natural or artificial membranes into nanoscale lipid-bilayer patches that closely mimic the lamellar organization of cellular membranes. Yet, this promising technology is limited by alterations in the conformational dynamics of embedded MPs and surrounding lipids by current polymer scaffolds. Two further limitations are the presence of UV-absorbing aromatic rings and the high charge density due to the polymer’s carboxylic groups.

NanoBelt aims at developing novel amphiphilic polymers that combine good membrane-solubilization yields with low UV absorption, negligible charge density, and—most importantly—maintenance of the function and conformational dynamics of encapsulated proteins. The rationale derives from the fact that the properties of membrane-solubilizing amphiphiles strongly depend on their molecular structure, monomer sequence, hydrophobicity, and molar mass distribution, which in turn modulate the conformational dynamics of proteins and lipids embedded in nanodiscs. These crucial variables cannot be unraveled and further improved unless systematic series of polymers with well-defined properties are made available.

Polymers will be prepared by Partner 1 (Inst. des Biomolécules Max Mousseron–Avignon University) either by post-functionalization of existing polymers with appropriate polar and apolar groups or by de novo synthesis of polymers from functionalized monomers. Partner 2 (Technische Universität Kaiserslautern) will characterize the potency of the new polymers to solubilize lipids and form nanodiscs as well as their ability to extract MPs from cellular membranes. The most promising of these polymers will then be selected for studying sensitive G protein-coupled receptors (GPCRs). The ghrelin receptor (GHSR) will be used as a prototypical class A GPCR by Partner 3 (Inst. des Biomolécules Max Mousseron–CNRS). To this end, the recombinant receptor will be assembled into nanodiscs formed with the new polymers, and its pharmacological properties will be evaluated and compared to existing methods. Partner 4 (Leipzig University) will use the corticotropin releasing factor receptor (CRF1R) as a model of class B GPCRs. With the aid of new FRET sensors, the conformational dynamics of these challenging drug targets will be dissected in the native-like but controlled lipid-bilayer environment offered by the novel polymer-based nanodiscs.

By using a systematic approach of validation based on the complementary skills of Partners 2, 3 and 4, NanoBelt aims at providing structure–activity relationships that will enable the rational design of improved polymers synthesized by Partner 1. The ultimate goal consists in identifying and establishing new polymers that extract MPs and MP complexes with their surrounding lipids directly from cellular membranes in a mild yet efficient manner to form polymer-encapsulated lipid-bilayer nanodiscs that retain the native structures and functions of the extracted proteins.